High prevalence and low intensity of Echinophthirius horridus infection in seals revealed by high effort sampling

Seal lice (Echinophthirius horridus) are bloodsucking ectoparasites of phocid seals and vectors of pathogens like the heartworm, Acanthocheilonema spirocauda. Grey and harbour seal populations are recovering in German waters and wildlife health surveillance is crucial for wildlife conservation. A new, high effort sampling protocol for seal lice was applied for grey and harbour seals along the German North- and Baltic Sea coast. Freshly dead seals were systematically sampled within a health monitoring of stranded seals over 12 months. Prevalence, intensity and distribution patterns of seal lice were analysed. 58% of harbour seals (n = 71) and 70% of grey seals (n = 10) were infected with seal lice. A majority of harbour seals displayed mild levels of infection, while three were moderately and two were severely infected. The head was the preferred predilection site, indicating that E. horridus prefers body areas with frequent access to atmospheric oxygen. Nits and different developmental stages were recorded in all age classes in grey and harbour seals in all seasons. For the first time, copulating specimens of E. horridus were recorded on a dead harbour seal, highlighting that E. horridus reproduces throughout the year on seals of all age classes in German waters.

sea lions (Otaria flavescens), which are habituated to the presence of humans 35 .Other methods involved restraining juvenile South American sea lions while combing the animal and collecting lice but without sampling the head area 36 .Quantitative lice collection on live pinnipeds remains logistically challenging and incomplete.Postmortem investigations however, are useful for recording a variety of health parameters, providing information about the cause of death [37][38][39][40] , including ectoparasites and associated lesions 24 .Patterns of louse distribution on their pinniped host contribute to understanding the adaptations of a long co-evolutionary relationship between parasite and host, allowing both parties to survive 41 .The detection of nits and developmental stages is crucial for decoding the reproductive strategies of this marine insect, adapted to reproduce on diving hosts, which can spend weeks or months at sea 42 .
E. horridus prevalence was previously investigated in the frame of post-mortem sampling in the North-and Baltic Sea 11,24,43 and in North American waters 44 .Although the seal louse prevalence in the Wadden Sea, Northern Europe was low after the Phocine Distemper Virus epidemics 11,24,43 , the prevalence at the Washington Coast, USA was 45% 44 .Ectoparasite records are often biased, as parasites typically leave their host actively after the death of their host 25,33 or are lost after death during drifting at sea, the stranding event, scavenging, or during transport and storage until the necropsy 11,24 .Intensity of E. horridus infections was recorded semi-quantitatively during post-mortem investigations 11,24 and distribution patterns of seal lice have not been systematically reported.Therefore quantitative data on seal lice infections are scarce, and little knowledge exists on their predilection sites on grey and harbour seals.The aim of this study was to systematically record prevalence and intensity of seal lice by applying a personnel-intensive strategy to reduce sampling bias in ectoparasite records and to obtain infection parameters as close to in vivo ectoparasite infection as possible.In this study, a novel post-mortem sampling strategy for E. horridus revealed new insights into seal louse prevalence, intensity and distribution patterns.

Study area and sampling
Seventy-one harbour seals and ten grey seals, collected within the stranding network of the federal state of Schleswig-Holstein (SH) along the North-and Baltic Sea coast from April 2022 until April 2023, underwent a newly developed, personnel-intensive examination.Seals included in this study were either found freshly dead or mercy killed by a licensed seal ranger.Carcasses were transported in a sealed plastic bag to the Institute for Terrestrial and Aquatic Wildlife Research (ITAW) in Büsum.One veterinarian systematically screened all animals for ectoparasites.Before examination, blood and organic contaminants were removed by washing with water to gain clear view of the fur.Body areas were categorised in body area 1: head, body area 2: fore or/and the hind flippers, tail, body area 3: head and fore and/or hind flippers, tail, and body area 4: complete body of the animal/ no clear distinction of infection possible.The seals were combed with a commercially available louse comb for human head lice (Dirk Rossmann GmbH, 2022).The direction of combing was parallel to the direction of the fur, which provided a smooth surface.The sleek fur coat of the carcass was then examined for small knoblike irregularities displaying the presence of seal lice (Fig. 1).All lice specimens, nits and developmental stages were removed with a louse comb or forceps and preserved either in ethanol (70%), formalin (10%) or were frozen.Nymph stages 1-3 were morphologically identified according to Leidenberger et al. 10 .Lice specimens were counted and the

Statistical analysis
Prevalence and intensity of infection were recorded according to Bush et al. 45 .Prevalence and statistical significance were calculated using the software Quantitative Parasitology (QPWeb, V 1.0.15) 46.To test the independent variables "sex" (male, female), "infected body area" (1-4) and "age groups" (AG1, AG2, AG3) in terms of significant differences in intensity (dependent variable) a Kruskal-Wallis test was performed.To determine in which groups differences in regard to the dependent variable can be observed, Dunn´s test with Bonferroni correction was performed, and the effect size was calculated by r = z sqrt(N) 47 .Significance level was set at 0.05.Statistical analysis regarding intensity was carried out using R 4.2.1(RCore Team, 2021) using the dplyr 48 , rstatix (v0.7.0; 49 and ggplot 2 (v 3.4.1; 50) packages.
70% (n = 7) of grey seals (n = 10) were infected with seal lice.Five out of six male grey seals were infected, while two out of four females were infected.
Two out of two young-of-the-year grey seals were infected with seal lice, one yearling (n = 3) and four of the adult grey seals (n = 5) were infected.No significant differences were observed in prevalence between different sexes (Fisher´s exact test, P = 0.5) or age groups (Fisher´s exact test, P = 0.33).

Reproduction of E. horridus
Nits and nymph stages 1-3 of E. horridus were found on harbour and grey seals and on males and female seals of all age classes.Nits and developmental stages occurred throughout the sampling year in all seasons (see Table 1, Supplementary Data 1).
In April 2023, two adult live lice were observed during copulation (Fig. 4) on a female yearling harbour seal from the North Sea, approximately 12 hours after the animal was mercy killed.The two specimens were subsequently preserved in ethanol (70%).The male louse was attached to the hair and skin of the seal´s head.The dorsal site of the abdomen of the male louse was placed underneath the ventral abdomen of the female louse.The abdomen of the male was dorsally bent with the male genitalia pointing towards the genital region of the female.The female louse was attached to the hair shafts of the seal.

Discussion
In this study, the prevalence of seal lice in harbour and grey seals in the North-and Baltic Sea are considerably higher than that reported in previous studies.In the same geographical study area, a notably lower prevalence of 3.4% in harbour seals was recorded between 1996 and 2013 11 , and 4% in harbour and 10% in grey seals were recorded between 2014 and 2021 24 based on post-mortem investigations.In 1971/72 a prevalence of 41% was recorded for mainly hunted harbour seals in the Wadden Sea in Lower Saxony, Germany 51 .No seal louse infection was observed in dead harbour seals from the Kattegat-Skagerrak and the Baltic region after the first Phocine www.nature.com/scientificreports/Distemper Virus epidemic 43 .Studies based on sampling of live animals in Scottish waters found a prevalence of 39% in the local harbour seal population 33 .
Results highlight standardized, detailed and comprehensive visual examination of freshly dead aquatic mammals to be an effective method to determine prevalence of permanent ectoparasites 25,26 , including the marine insect E. horridus.The striking difference with regard to previous post-mortem studies of the North-and Baltic Sea presumably depends on the high effort sampling protocol.In the present study, only freshly dead animals were examined, and loss of ectoparasites due to scavengers, drifting at sea, storage and freezing of the carcass was limited.Systematic and detailed screening carried out by trained personnel is crucial to detect seal lice, which are well camouflaged and remain strongly attached to their host.Nevertheless, possible post-mortem migration of parasites could influence the observed distribution patterns and intensity.While ectoparasites are known to quickly leave their dying host 25,52 , in small terrestrial mammals, lice were observed to leave their host later than ticks and fleas 53 .The results of this study contradict previous assumptions that seal lice might leave their host immediately after its death and remain in the haul-out substrate 33 .Rather, this indicates that seal lice stay on their host, unless another suitable host is in accessible proximity.Sick or weak animals are often unable to return to the water after haul-out sessions 54 , hence remain isolated on land 55 ; consequently, lice are forced to remain on their host.Additionally, in weakened host individuals self-grooming might be limited and thus the ectoparasite load increases 56,57 .However, recovering harbour and grey seal populations and higher densities of seals on shared haul-out sites may also facilitate interspecies and intraspecies transmission and higher prevalence 24 .
This study highlights that seal louse prevalence in harbour and grey seal populations in the North-and Baltic Sea is much higher than previously assumed.Seal louse infections are widespread, but levels of infection are predominantly mild and limited to few parasite individuals per host.This aggregated distribution of parasites in natural host populations has been observed for many parasites 58,59 , most host individuals display a mild or no parasitic infection, while only a few host individuals are severely parasitized.
The possible role of E. horrdius as vector of A. spirocauda remains a central object regarding the importance of seal louse prevalence.Microfilaria were found in blood smears of 41% of wild-caught seals sampled from   11 , and the present study reportes similarly high prevalence of E. horridus in the same geographical area.This finding supports E. horridus as possible vector for the filarial heartworm, A. spirocauda 24 .Contrary, prevalence of adult heartworms located in the heart has been considerably lower 11,24 .The death of adult female nematodes after the release of microfilarial stages into the blood, called ephemerality, is common in other filarioid nematodes, in which production of microfilaria is minimized to a certain amount of time, preventing vectors from continuously ingesting microfilaria 60 .In this study, the head was the body area most often infected with seal lice.In all observed cases, in which only the head area was infected, the level of infection was mild.If animals were severely infected, the entire body was infected.Results indicate the head as preferred body area, while the rest of the body is parasitized when the space around the head becomes limited due to increasing population size of the parasite on its host.Distribution patterns of ectoparasites on hosts are mainly determined by the microhabitat most suitable for the parasite, e.g. in terms of grooming and differing skin thickness 61 .In birds, lice parasitize areas of the body that are difficult for the animal to reach while preening 25,62 .E. horridus has also been suggested to infect body parts difficult to access for their host 63 .Due to the anatomical characteristics like highly specialized and motile fore flippers 64,65 , seals are able to groom all parts of their head.During movement on land, the ventral abdomen and flippers are mechanically stressed, which could prevent ectoparasites from infecting those areas 16 .During periods at sea, the seal´s head is most frequently above the water surface and in contact with atmospheric oxygen, crucial for seal lice respiration.For Antarctophthirus callorhini, and Proechinophthirus fluctus, seal lice found on Northern Fur Seals (Callorhinus ursinus), surface temperatures of 25-35 °C were considered optimal for development of the larval stages 17 .In harbour seals, the surface temperature of the head ranges around 15 °C, while the snout in particular accomplishes surface temperatures up to 20 °C66 .The high vascularization of the sensory tactile system 67 and the reduced blubber thickness in the head area 68 could additionally benefit the feeding process of the hematophagous insect, providing a small distance between body surface and vessels, allowing seal lice to feed and to complete their lifecycle.
In particular in juveniles, infection of the head area could be an indication of the transmission pathway of E. horridus.The harbour seal mother keeps nose-to-nose contact continuously within the first minutes postpartum 69 .Nose-to-nose, nose-to-body and contact during suckling are maintained in the following weeks during nursing 70 , thus the head could be the area most likely to be infected first.For other seal lice species, infections of pups are observed hours after birth 14,17 .It should be noted that E. horridus was sampled from adult harbour seals but the sample size remains low compared to other age classes.
In single cases, infections of the head area with E. horridus 71,72 were described, while severe levels of infection were present on the ventral surface of neck, sacral and genital regions in harbour seals 73 .A systematic study based on E. horridus infected live harbour seals on Scottish shores suggested that the primary infection pattern was the hind flippers 33 , similar to infection patterns found in otariid seals 32,74 , although the entire body was not sampled.Higher prevalence of eggs and ovigerous female lice (Antarctophthirus microchir) on the dorsal back compared to the ventral belly was reported for South American Sea lion pups (O.flavescens), while for nymphs and male A. microchir, the opposite pattern was recorded.However, only limited conclusions are possible as the head, neck and fore flippers were not examined 16 .
Data on the reproductive cycle of E. horridus is scarce.In this study, nits were found on young-of-the-year harbour seals as well as on yearling seals in all seasons.In grey seals, nits and nymphal stages were sampled from a yearling grey seal and two adult grey seals.Seal lice reproduced on a yearling harbour seal in April.Findings of the current study indicate that E. horridus does reproduce independently of grey and harbour seal reproductive behaviour.Contrary to this finding, it was assumed that seal lice species Antarctophthirus microchir on South American sea lions (O.flavescens) synchronize their reproduction with that of their host 14,75,76 .On pups, which spend sufficient time on land, seal lice are able to reproduce and complete their nymphal development 16,77,78 .Findings from this study contrast with reports of seal lice infecting pinnipeds in the Southern hemisphere (e.g. A. microchir) which are suggested to produce only one to two lice generations per year during the pupping season of their host 14,77 .In the Northern hemisphere, E. horridus infects two phocid seal species with different parturition characteristics and lactation times.While harbour seals give birth from May to August, grey seals are born during winter (November-February) 79 .While harbour seals shed their fetal pelage prenatally, grey seal pups are born with lanugo and undergo their first moult approximately 3-6 weeks post-partum 79 .Harbour seal pups are able to swim with their mothers within hours after birth 80 , while grey seal pups remain on land for their lactation period of approximately 19 days and in some populations even during their fasting period after weaning 81 .When E. horridus is transmitted to a grey seal pup, it needs to survive the impediment of the lanugo fur moult, but the time on land is extended compared to that of harbour seal pups.Our results highlight that the ectoparasitic insect E. horridus has adapted to different and challenging reproductive behaviours of two seal species and has evolved to complete its life cycle successfully on a marine host.

Conclusion
Novel quantitative data about seal lice prevalence, intensity, and infection patterns are reported for two phocid hosts.New information about nits and developmental stages of seal lice on grey and harbour seals was collected from all age classes and the entire seal body for the first time.The findings underline the importance of performing profound and systematic post-mortem investigations within a fast and efficient marine mammal stranding network, essentially relying on quick communication between wildlife researchers and seal rangers and other non-scientific helpers.Ectoparasites represent an important indication of ecological relationships, highlighted by increasing prevalence of E. horridus corresponding with recovering population sizes of harbour and grey seals.Higher seal numbers on haul-out sites enable inter-and intraspecific ectoparasite transmissions.Seal lice

Figure 1 .
Figure 1.Different infected body parts in juvenile harbour seals.(a) E. horridus located close to the eyelid.(b) E. horridus infection in the muzzle area.(c) E. horridus infection in abdominal region.(d) Close-up image of live E. horridus moving on harbour seal fur.(e) E. horridus located on fore flipper, between harbour seal claws.(f) E. horridus infection of hind flipper.a-f, White arrows pointing at E. horridus.

Figure 2 .
Figure 2. Intensity of E. horridus infection in different groups.(a) Intensity of infection with E. horridus in harbour and grey seals from the North-and Baltic Sea (from April 2022 until April 2023).(b) Intensity of infection in different age groups of harbour seals.

Figure 3 .
Figure 3. Levels of E. horridus infection and distribution patterns in grey and harbour seals.(a) Infected body areas 1-4 and levels of infection in harbour seals from the North-and Baltic Sea (from April 2022 until April 2023).(b) Infected body areas 1 and 4 and levels of infection in grey seals from the North-and Baltic Sea (from April 2022-April 2023).Pictograms represent the species and dark-grey shaded areas symbolize infected body areas.

Figure 4 .
Figure 4. Reproduction of E. horridus.(a) Female and male E. horridus during copulation on a yearling harbour seal.(b) Close-up of male genitalia.a, b: bars: 1 mm.

Table 1 .
Nits and developmental stages of E. horridus on grey and harbour seals recorded from April 2022 until April 2023.Symbols explained: + = present; − = absent.

Marine mammal species Age group Sex Sampling month Level of infection Nit Nymph 1 Nymph 2 Nymph 3
as potential vectors for multiple pathogens need to be monitored with regard to environmental changes, which can accelerate host-parasite interactions and facilitate ectoparasite transmission.